PAH ACTION ON OVARIAN STEROIDOGENESIS IN VITRO

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DESCRIPTION: (adapted from the applicant's description) The general hypothesis of this proposal is that polycyclic aromatic hydrocarbons (PAH) are endocrine-disrupting chemicals that exert their untoward cellular effects on female reproductive health by a direct action at the level of the ovary via impairing ovarian follicular health through cellular degeneration (atresia)/programmed cell death (apoptosis) and by altering steroid synthetic capability. It is proposed that such environmental xenobiotics exert these effects by inducing an increased incidence of apoptosis in follicular granulosa cells (GC); and affect steroidogenesis at one or more of several loci in the biosynthetic pathway: at P450 side-chain cleavage, 3b-hydroxysteroid dehydrogenation, P45017,20 lyase cleavage, and/or at the level of aromatization. TCDD and another AH receptor (AHR) agonist with lesser affinity, beta-naphthoflavone (BNF), will be administered in organoid tissue culture to intact ovarian follicles from adult cycling Holtzman rats and also to primary cell cultures of human granulosa cells retrieved from women undergoing in vitro fertilization procedures; and effects on incidence of apoptosis and steroidogenesis evaluated. Specifically, the investigators will characterize the effects of AHR agonists on (A) the detection of apoptosis in situ using immunofluorescence localization of DNA fragmented internucleosomally; (B) localize cytochemically the enzymes involved in and analyze biochemically/radiometrically the conversion of rat thecal mitochondrial cholesterol to pregnenolone (P5); (C) the conversion of P5 to progesterone (P4) in smooth endoplasmic reticulum; (D) the microsomal cleavage of P5 to dehydroepiandrosterone; and (E) the aromatization of androstenedione to estrone in rat follicles and human granulosa cells. The investigators will correlate morphologic changes and alterations in biosynthesis that characterize toxin-induced demise of ovarian follicles.